Not applicable.
This invention relates generally to methods of using modified silica matrices to clear solutions of disrupted biological material, such as cell lysates or homogenates of plant or animal tissue. This invention also relates to the use of such matrices to isolate target nucleic acids, such as plasmid DNA, chromosomal DNA, DNA fragments, total RNA, mRNA, or RNA/DNA hybrids from non-target material, such as proteins, lipids, cellular debris, and non-target nucleic acids. This invention relates, particularly, to the use of silanized silica matrices in lysate clearance and in target nucleic acid isolation.
Various methods have been developed for isolating target nucleic acids from biological material or from other types of material containing the nucleic acids. When the target nucleic acid is contained in the interior of a cell, the cell membrane must be disrupted and the contents of the cell released into the solution surrounding the cell before the target nucleic acid can be isolated from other cellular material. Such disruption can be accomplished by mechanical means (e.g., by sonication or by blending in a mixer), by enzymatic digestion (e.g., by digestion with proteases), or by chemical means (e.g., by alkaline lysis followed by addition of a neutralization solution). Whatever means is used to disrupt a cell, the end product, referred to herein as a lysate solution, consists or the target material and many contaminants, including cell debris.
Centrifugation or filtration are commonly used to clear a lysate solution of as many of the large contaminants as possible before the target nucleic acid material is isolated therefrom. Unfortunately, neither filtration nor centrifugation is readily amenable to automation. Specifically, neither are typically performed at basic pipettor-diluter robotics stations, such as the Biomek(copyright)-2000 (Beckman Coulter, Inc.; Fullerton, Calif.).
Many materials and methods have been developed for use in the isolation of nucleic acids from cleared lysate solutions. One such method is extraction of a nucleic acid from an agarose gel after fractionation of the nucleic acid by gel electrophoresis. Known means of extraction of nucleic acids from gel slices include dialysis, solvent extraction, and enzymatic digestion of the agarose. Such systems of nucleic acid extraction from an agarose gel slice tend to be very labor-intensive, and not amenable to automation. Furthermore, smaller sized fragments of DNA or RNA (i.e., below about 100 base pairs) tend to be lost in the extraction process.
Other systems of nucleic acid extraction are silica based, such as those which employ controlled pore glass, filters embedded with silica particles, silica gel particles, resins comprising silica in the form of diatomaceous earth, glass fibers or mixtures of the above. Each such silica-based solid phase separation system is configured to reversibly bind nucleic acid materials when placed in contact with a medium containing such materials in the presence of chaotropic agents. The silica-based solid phases are designed to remain bound to the nucleic acid material while the solid phase is exposed to an external force such as centrifugation or vacuum filtration to separate the matrix and nucleic acid material bound thereto from the remaining media components. The nucleic acid material is then eluted from the solid phase by exposing the solid phase to an elution solution, such as water or an elution buffer. Numerous commercial sources offer silica-based resins designed for use in centrifugation and/or filtration isolation systems, e.g., Wizard(copyright) DNA purification systems products from Promega Corporation (Madison, Wis., U.S.A.), or the QiaPrep(copyright) DNA isolation systems from Qiagen Corp. (Chatsworth, Calif., U.S.A.). Unfortunately, the type of silica-based solid phases described above all require one to use centrifugation or filtration to perform the various isolation steps in each method, limiting the utility of such solid phases in automated systems.
Magnetically responsive solid phases, such as paramagnetic or superparamagnetic particles, offer an advantage not offered by any of the silica-based solid phases described above. Such particles could he separated from a solution by turning on and off a magnetic force field, by moving a container on to and off of a magnetic separator, or by moving a magnetic separator on to and off of a container. Such activities would be readily adaptable to automation.
Magnetically responsive particles have been developed for use in the isolation of nucleic acids by the direct reversible adsorption of nucleic acids to the particles. See, e.g., silica gel-based porous particles designed to reversibly bind directly to DNA, such as MagneSil(trademark) Paramagnetic Particles (Promega), or BioMag(copyright) Paramagnetic Beads (Polysciences, Warrington, Pa., U.S.A.). See also U.S. Pat. No. 6,027,945. Magnetically responsive glass beads of a controlled pore size have also been developed for the isolation of nucleic acids. See, e.g. Magnetic Porous Glass (MPG) particles from CPG, Inc. (Lincoln Park, N.J. U.S.A.), or porous magnetic glass particles described in U.S. Pat. Nos. 4,395,271; 4,233,169, or 4,297,337. Nucleic acid material tends to bind very tightly to glass, however, so that it can be difficult to remove nucleic acids from such magnetic glass particles, once bound thereto. As a result, elution efficiencies from magnetic glass particles tend to be low compared to elution efficiencies from particles containing lower amounts of a nucleic acid binding material such as silica.
A variety of silica matrices have also been developed which consist of a silica solid phase with ligands covalently attached thereto designed to participate in ion exchange or in reversed-phase interaction with nucleic acids. However, such systems are generally designed for use as a solid phase of a liquid chromatography system, for use in a filtration system, or for use with centrifugation to separate the solid phase from various solutions. Such systems range in complexity from a single species of ligand covalently attached to the surface of a filter, as in DEAE modified filters (e.g., CONCERT(copyright) isolation system. Life Technology Inc., Gaithersburg, Md., U.S.A.), to a column containing two different solid phases separated by a porous divider (e.g., U.S. Pat. No. 5,660,984), to a chromatography resin with pH dependent ionizable ligands covalently attached thereto (e.g., U.S. Pat. No. 5,652,348), to mixed-mode or mixed-bed resins with ion exchange ligands and reversed-phase ligands on the same or on different solid phase components of the resins, respectively (e.g., McLaughlin, L. M., Chem Rev (1989) 89:309-319).
Matrices have also been developed which are designed to reversibly bind to specific target materials through affinity interaction. Some such matrices use affinity of the poly (A) tail of mRNA for oligo (dT) to isolate mRNA, either by attaching oligo (dT) directly to the surface of a solid phase (e.g., U.S. Pat. No. 5,610,274), or by providing a solid phase coated with streptavidin and biotinylated oligo (dT) which naturally binds to both the streptavidin and to mRNA in a solution (e.g., PolyATract(copyright) Series 9600(trademark) mRNA Isolation System from Promega Corporation (Madison, Wis., U.S.A.); and ProActive(copyright) streptavidin coated microsphere particles from Bangs Laboratories (Carmel, Ind., U.S.A.)).
Silanization has been used as a coupling agent to facilitate the covalent attachment of various ligands to the silica solid phases to produce chromatographic matrices for the isolation of solutes, such as nucleic acids. See, e.g., U.S. Pat. No. 4,672,040 (col. 13, lines 3-22); U.S. Pat. Nos. 5,734,020; 4,695,392; and 5,610,274). In such reactions a silane compound, such as 3-glycidoxypropyltrimethoxysilane, is reacted with the surface of a solid phase, such as a silica based material or with an iron oxide, such that the silane becomes attached thereto. The resulting matrix includes highly reactive residues, such as the epoxide group at the terminus of the alkoxy chain of 3-glycidoxypropyltrimethoxysilane, which remain available after reaction with the surface of the solid phase. Matrices with such reactive groups have been used as intermediaries in the production of ion exchange, reversed-phase, mixed-bed, mixed-mode, and affinity resins.
Each of the systems described above has its limitations in terms of solution conditions required for the isolation of nucleic acids, and in terms of the class of substrates from which it can best isolate nucleic acids. To date, no materials and methods have been developed which can be used to isolate low molecular weight nucleic acids, (e.g., under 100 base pairs) as efficiently as higher molecular weight nucleic acids from substrates such as vegetable oils or agarose gel slices. With the modern need to detect viruses and evidence of genetic modification of vegetable matter from which such oils are produced, there is a great need to be able to isolate small quantities of nucleic acids from such substrates. Materials and methods are also needed which enable one to automate as many steps as possible to quickly and efficiently isolate target nucleic acids from cells or mammalian tissue. Specifically, methods and materials are needed for the clearing of solutions of disrupted biological material, and for the isolation of target nucleic acids from such cleared solutions, wherein labor-intensive steps such as filtration or centrifugation are not required. The present invention addresses each of these needs.
The method of the present invention provides a means for clearing a solution of disrupted biological material, using a first silica solid phase with a plurality of first silane ligands covalently attached thereto, wherein the first silane ligands are designed to adsorb to non-target nucleic acid in the disrupted biological material. The method of the present invention also provides a means for isolating a target nucleic acid from a solution, such as a cleared lysate solution, comprising the target nucleic acid and at least one non-target material using a second silica solid phase with a plurality of second silane ligands covalently attached thereto, wherein the second silane ligands are designed to adsorb selectively to the target nucleic acid in a nucleic acid adsorption solution. In a preferred embodiment of the method of the present invention, a solution of disrupted biological material is cleared with the first silica solid phase in the first solution, and target nucleic acid material removed from the resulting cleared lysate by adsorption to the second silica solid phase in a nucleic acid adsorption solution.
In the first embodiment of the present method described above, a solution of disrupted biological material, such as a cell lysate or a homogenate of mammalian tissue, is cleared according to steps comprising: (a) providing a first silanized silica matrix, comprising a silica solid phase with a plurality of silane ligands covalently attached thereto, and (b) combining the first silanized silica matrix with a first solution, comprising a disrupted biological material, a target nucleic acid, and a chaotropic salt concentration sufficiently high to promote selective adsorption of the disrupted biological material to the matrix, thereby forming a first complex. The disrupted biological material is preferably introduced to the matrix in the presence of a chaotropic salt concentration sufficiently high to promote selective adsorption of the disrupted non-target biological material to the matrix, leaving the target nucleic acid in solution.
In the second embodiment or the method of the present invention described above, a target nucleic acid is isolated from a second solution, a nucleic acid adsorption solution comprising the target nucleic acid and at least one non-target material. The second solution can be from any one of a number of different sources including, but not limited to, a cleared lysate, a vegetable oil, a cleared homogenate of mammalian tissue, a cleared lysate of vegetable material, or a solution comprising a target nucleic acid fractionated by gel electrophoresis in an agarose gel. This embodiment of the method comprises the steps of: (a) providing a second silanized silica matrix comprising a silica solid phase with a plurality of silane ligands of general formula (I), below, covalently attached thereto; (b) combining the second silanized silica matrix with the nucleic acid adsorption solution, wherein the pH of the nucleic acid adsorption solution is less than about pH 8.0 and the concentration of chaotropic salt in the solution is sufficiently low that the target nucleic acid is selectively adsorbed to the second silanized silica matrix, thereby forming a second complex.
Each of the plurality of silane ligands of the first and second silanized silica matrix is preferably of the general formula (I): 
wherein R1 and R2 are each a subunit selected from the group consisting of a hydrocarbon chain having from 1 to 5 carbon atoms, an alkoxy having from 1 to 5 carbon atoms, a hydroxyl, an alkyl chain having from 4 to 10 carbon atoms interrupted by an oxy residue wherein up to five of the carbon atoms is substituted by a group selected from the group consisting of a halogen, an alkoxy having from 1 to 3 carbon atoms, a cyano having from 1 to 3 carbon atoms, and a hydroxy; wherein R3 is a hydrocarbon chain having from 1 to 20 carbon atoms substituted by at least one hydroxy, an alkyl chain having from 4 to 20 carbon atoms interrupted by at least one oxy group wherein up to ten carbon atoms are replaced by a moiety selected from the group consisting of a halogen, a cyano having from 1 to 3 carbon atoms, an alkoxy having from 1 to 3 carbon atoms, a hydroxy, and an epoxy. R1 and R2 may also independently be linkages to other silane ligands to generate higher order polymers.
In yet another embodiment, the present invention consists of a kit comprising, in a single container, a plurality of silanized silica magnetic particles comprising a silica solid phase with a plurality of silane ligands covalently attached to the surface of each particle, each ligand having a structure of formula (I), above.
The methods and materials of the present invention can be used to isolate target nucleic acids including, but not limited to, plasmid DNA, DNA fragments, total RNA, mRNA, RNA/DNA hybrids, amplified nucleic acids, and genomic DNA from a variety of contaminants, including but not limited to agarose from an agarose gel, non-target nucleic acids, and non-target components of bacteria, animal tissue, blood cells, and vegetable oil or other plant material. The methods and materials of the present invention are efficient at isolating both low molecular weight DNA molecules (i.e., less than about 150 base pairs) and larger molecular weight DNA. Applications of the methods and compositions of the present invention to isolate nucleic acids from a variety of different media will become apparent from the detailed description of the invention below. Those skilled in the art of this invention will appreciate that the detailed description of the invention is meant to be exemplary only and should not be viewed as limiting the scope of the invention.